Journal
Journal of Applied Horticulture, 15(2): 71-80, 2013
Appl
Molecular biology of Tomato spotted wilt virus: An update
Saurabh Kulshrestha*, Anshul Sharma and Chandrika Attri Seth
Faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Bajhol, Solan,
Himachal Pradesh, India. *E-mail: saurabh_kul2000@yahoo.co.in
Abstract
Advances in understanding of Tomato spotted wilt virus (TSWV) molecular biology are reviewed. TSWV, a type species of the genus
Tospovirus, is an enveloped virus that causes high economical losses in many crops worldwide. It is transmitted by several species
of thrips and multiplies in insect cells. The most important vector is Frankliniella occidentalis which transmits TSWV in a persistent
propagative manner. Several factors are known from both virus and vector side which plays important role in virus acquisition by thrips
and its subsequent transfer. TSWV is a segmented negatively strand RNA virus. RNA of TSWV is partitioned among three negative
or ambisense single stranded RNA (ssRNA) labeled as L, M and S in order of decreasing size, (approximately 8897, 4821 and 2916
nucleotides long, respectively). These RNA segments encode various proteins like N and Ns by S RNA; NSm and G1/G2 by M RNA
and RdRp by L RNA. Intergenic region present in M and S RNA of TSWV helps in proper transcription of different genes encoded by
M and S RNA. The different proteins encoded by TSWV genome help the virus in protection, cellular movements, vector transmission,
replication and recently in RNA silencing suppressor activity. The present review focuses on basic structure, genome organization,
molecular basis of transmission and recent advances in TSWV detection.
Key words: TSWV, Thrips, L RNA, M RNA, S RNA, Intergenic region
Abbreviations: AMV=Alfalfa mosaic virus; ArMV= Arabis mosaic virus; CERV= Carnation etched ring virus; cDNA= complementary
DNA; DAS ELISA = double antibody sandwich enzyme linked immunosorbent assay; ds = double stranded; ELISA = enzyme linked
immunosorbent assay; FoTf = Frankliniella occidentalis putative transcription factor; GA = Georgia; GFP = green fluorescent protein;
GP = glycoprotein precursor; IGR = intergenic regions; IRES = internal ribosomal entry site; FL = Florida; FRET = fluorescence
resonance energy transfer; FLIM = fluorescence lifetime imaging microscopy; miRNA = micro-RNA; MP = movement protein; NC =
nucleocapsid; ORF = open reading frame; PNGase F = peptide:N-glycosidase F; PTGS = post transcriptional gene silencing; PNRSV
= Prunus necrotic ring spot virus; PCR = polymerase chain reaction; RNPs = ribonucletidproteins; RdRp = RNA dependant RNA
polymerase; RT-PCR = real time polymerase chain reaction; siRNA = small interfering RNA; ss = single stranded; SLRSV = Strawberry
latent ringspot virus; SDS-PAGE = sodium dodecyl sulphate- polyacrylamide gel electrophoresis; TSWV = Tomato spotted wilt virus;
TMMV = Tuberose mild mottle virus; UTR = untranslated region; vc = virus complementary; WFT = western flower thrips
Introduction
Tomato spotted wilt virus (TSWV) is found in almost all continents
and is the type species of the plant viruses belonging to the genus
Tospovirus within the arthropod-borne Bunyaviridae family (Van
Regenmortel et al., 2000). TSWV is one of the most economically
important members of the genus Tospovirus (Peters and Goldbach,
1995; Moyer, 2000). From family Bunyaviridae, genus Tospovirus
is the only genus consisting of viruses infecting plants (Pappu,
2008). TSWV is transmitted by multiple species of thrips (Ullman,
1997) and the most important vector is Frankliniella occidentalis
Pergande, the western flower thrips (WFT) which transmits TSWV
in a persistent propagative fashion (Gera et al., 2000). It was not
until mid-1960 that the enveloped virions morphology was revealed
and the molecular characterization and genome organization did not
occur until after 1990 (Moyer, 1999).
It seems that disease caused by the TSWV was first observed in
1906 (Sakimura, 1962). Brittlebank (1919) published the first
description of this new disease, detected on tomatoes in 1915
in the state of Victoria (Australia) and he called it “spotted wilt
of tomato”. In the same year, Osborn also observed this disease
on tomatoes in the south of Australia and in 1920 its presence
was reported in all the Australian states (Best, 1968). The first
characterization of this virus as the causal agent of the disease
was reported by Samuel et al. (1930), who gave it its current
name “Tomato spotted wilt virus”. Since then it was reported
from several tropical and temperate regions and it is considered
worldwide in distribution. Worldwide loss caused due to this virus,
mainly of commercial vegetable crops, is around one billion dollars
annually (Scott, 2000). TSWV ranks among the top 10 of the most
detrimental plant viruses worldwide (Prins and Goldbach, 1998).
In general, the two main concomitant factors that make TSWV
one of the most destructive and widely distributed plant viruses
are the highly polyphagous nature of its vectors and the relative
lack of host specificity of the virus. Consequently, TSWV has
the widest host ranges of any known plant viruses and its control
remains problematic. Major crops susceptible to TSWV infection
are tomato, pepper, lettuce, potato, papaya, peanut, tobacco and
chrysanthemum (German et al., 1992). Symptoms vary with the
host plant, time of year and environmental conditions and include
stunting, necrosis, chlorosis, ring spots and ring/line patterns
affecting leaves, stems and fruit (German et al., 1992; Mumford et
al., 1996). The current list of TSWV hosts consists of 1090 plants
species belonging to 15 families of monocotyledonous plants, 69
families of dicotyledonous plants and one family of pteridophytes
(Parrella et al., 2003).
72
Molecular biology of Tomato spotted wilt virus: An update
Basic Structure: TSWV particles can be readily detected by
electron microscope observations of leaf dip preparations with
or without gold-immunolabelling (Milne, 1993). With the help
of electron microscope, it has been shown that TSWV virions are
spherical shaped (between 80 to 110 nm diameter). Each viral
particle consists of granular core bounded by an envelope with
surface projections (Best and Palk, 1964; Ie, 1964; Martin,1964;
Kitajima, 1965). Like the other bunyaviruses, membrane
envelope of TSWV contains virally encoded glycoproteins, a
feature quite uncommon for plant-infecting viruses but rather
typical among animal viruses. Virion composition is 5% nucleic
acid (RNA), 70% protein, 5% carbohydrate, and 20% lipid. Very
little information was available concerning the nucleic acid of
TSWV till 1965. Later, it was concluded that the nucleic acid was
RNA on the basis of positive orcinol and negative diphenylamine
reactions (Van Kammen et al., 1966). Best (1968) arrived at the
same conclusion from data obtained by paper chromatography
and estimation of the base composition of the nucleic acid
(adenine 35 %, cytosine 9 %, guanine 38 % and uracil 18 %).
Genome organization
Tomato spotted wilt virus (TSWV) is an enveloped segmented
negatively strand RNA virus. Negative strand RNA viruses are
also known as antisense strand RNA viruses. The term ‘negative
strand viruses’ is derived from the coding strategy adopted by
segments of viral genomes. RNA molecules of negative strand
RNA viruses are transcribed first to give positive coding strand.
So, in negative strand RNA viruses during amplification cycle,
transcription is the first step (Van knippenberg, 2005). This
explains the presence of viral polymerase inside virus particle
(Van Poelwijk et al., 1993). In contrast, RNA molecule is of
coding or sense polarity (mRNA) in positive strand RNA viruses.
This RNA molecule can be directly utilized for the synthesis of
proteins if provided with a 5′ cap structure or internal ribosomal
entry site (IRES) (Van knippenberg, 2005). Within the group
of segmented negative sense RNA viruses, a distinct sub-group
is formed by the so-called ambisense viruses. These possess at
least one genome segment that is of dual polarity, i.e. containing
one reading frame in the viral-sense RNA and one in the viralcomplementary (vc) RNA. The Tospovirus genome is a tripartite,
partitioned among three negative or ambisense single-stranded
RNA (ssRNA) segments named L, M, and S in order of decreasing
size (Fig. 1) (de Haan et al., 1989). In addition, both the M RNA
and S RNA possess the characteristic A-U rich intergenic regions
(IGR) capable of forming stable hairpin structure (de Haan et al.,
1990). A conserved sequence found at the top of the hairpins of
TSWV S and M RNAs indicates a possible role of amino acid
signal for transcription termination of the open reading frames
(ORFs) within the ambisense RNA segments (de Haan et al., 1990;
Kormelink et al., 1992a). The IGR of M RNA is smaller in length
compared to the IGR of S RNA (Bhatt et al., 1999). Recently, it
has been studied that hairpin structure sequence of S ambisense
RNA in 3´ untranslated region (UTR) of mRNA plays important
role in translation also (Geerts-Dimitriadou et al., 2012). The
RNAs have partially complementary terminal sequences. This
complementarity for each individual segment is extended to ~ 65
nt, that allows the RNAs to adopt a pseudocircular or panhandle
conformation (de Haan et al., 1989, 1990; Kormelink et al.,
1992a; Elliott et al., 2000). The 5′ and 3′ terminal 8 nucleotides
(nt) of each genomic RNA segment are complementary and
conserved: 5′-AGAGCAAU and 3′-UCUCGUUA. The assembly
Fig. 1. Replication strategy for tripartite genome of TSWV (data taken from de Aliva, 1992; de Haan et al. 1990; 1991; Kormelink et al., 1992c;
Roselló et al., 1996)
Molecular biology of Tomato spotted wilt virus: An update
of virus particles first requires the formation of ribonucleoproteins
(RNPs). These RNPs are complexes of viral genomic RNA, N
protein and a few copies of the L protein (Elliott, 1990). The RNPs
are enclosed by a lipid membrane envelope, which contains the
viral-encoded glycoproteins. The viral genome contains five open
reading frames coding for six mature proteins (Table 1).
L RNA: The largest RNA (L RNA) is negative sense and
monocistronic. L RNA serves as a multifunctional, replicationassociated protein and is believed to function cooperatively with
host-encoded factors. L RNA is approximately 9 kb and has a
single open reading frame (ORF) in the viral complementary
sense. The RNA is 8897 nucleotides long coding for a 330 kDa
protein, which is the putative RNA-dependent RNA polymerase
(RdRp) or L protein (de Haan et al., 1991; Lee et al., 2011). This
330-kDa protein has been implied in several enzymatic activities
such as transcriptase, replicase and endonuclease (Adkins et
al., 1995; Van Poelwijk, 1996; Chapman et al., 2003). RNA
viruses show extremely high mutation rates, because of lack of
proofreading ability of their polymerases (Moya et al., 2000).
Although, the mutation or error rate of viral RdRp has not been
estimated for plant viruses, it has been measured for animal RNA
viruses and it is approximately 10–4, or one error per genome
per replication cycle (Roossinck, 1997). As Tospovirus has both
negative and ambisense coding strategies, the RNA dependent
RNA polymerase has to be co-transported with the viral RNA to
allow transcription and replication within the newly infected cells
(Soellick et al., 2000).
M RNA: The M RNA is approximately 4.8 kb (Lee et al.,
2011) and encodes a 34 kDa protein in viral sense designated
nonstructural NSm (commonly called as movement protein)
proposed to be involved in cell to cell movement of nonenveloped
ribonucleocapsid structures (Storms et al., 1995; Soellick et al.,
2000; Silva et al., 2001) and stimulation of tubule formation
in protoplasts. Viral movement proteins facilitate transport of
infectious material through plasmodesmata, the intercellular
connection for the plant cell (Griffiths et al., 1992). In plant
tissue, the NSm protein of TSWV functions as viral movement
protein (MP), aggregating into plasmodesma-penetrating
tubules to establish cell-to-cell movement. Upon heterologous
expression NSm was able to form similar tubules on the surface
of insect (Spodoptera frugiperda) cells, during expression and
cellular manifestation of this protein in infected thrips tissue. It
is shown that NSm, though detectably expressed in both the L2
larval and adult thrips stages, does not aggregate into tubules,
indicating that this requirement is associated to its function as
MP in plants, and raising the question if NSm has a function at
all during the insect life cycle of TSWV (Storms et al., 2002).
TSWV NSm domains required for tubule formation, movement
73
and symptoms were identified previously by deletion-mapping
and alanine-substitution mutagenesis using the TMV-based
system. Mutagenesis studies of TSWV NSm amino acids that
are conserved in other tospovirus were conducted by Li et al.
(2010) and suggested that functional domains of NSm protein
may be conserved across the genus. Recent findings suggest that
these movement proteins, which recognize and transport the viral
genomes as naked nucleic acid or in complex with other viral
proteins, resemble plant proteins that are involved in selective
trafficking of protein and protein- nucleic acid complexes through
plasmodesmata as part of fundamental transport and signaling
process (de Haan et al., 1990). The molecular basis of NSm
function was studied by expressing the protein in Escherichia coli
and investigated protein-protein and protein-RNA interactions of
NSm protein in vitro. NSm specifically interacts with TSWV N
protein and binds single-stranded RNA in a sequence-nonspecific
manner. Using NSm as bait in a yeast two-hybrid screen, two
homologous NSm-binding proteins of the DnaJ family from
Nicotiana tabacum and Arabidopsis thaliana were identified
(Soellick et al., 2000).
The viral complementary sense (vc RNA) ORF codes for
glycoprotein precursor GP, is post-translationally cleaved
into the spikes or glycoproteins G1 and G2 (Kormelink et
al., 1992a). For TSWV, the 58 kDa G2 is also referred to as
G(N) and 78 kDa G1 as G(C) (N and C refer to the amino and
carboxy terminal position within the glycoprotein precursor,
respectively) (Snippe et al., 2007). These glycoproteins are
required for virus infection of the arthropod vector. Other
members of the Bunyaviridae enter host cells by pH-dependent
endocytosis. During this process, the glycoproteins are exposed
to conditions of acidic pH within endocytic vesicles causing the
G(C) protein to change its conformation. This conformational
change renders G(C) more sensitive to protease cleavage.
TSWV virions were subjected to varying pH conditions and
determined that TSWV G(C), but not G(N), was cleaved under
acidic pH conditions and this phenomenon was not observed
at neutral or alkaline pH. This provides evidence at low pH,
G(C) conformation changes, which results in altered protease
sensitivity. Furthermore, sequence analysis of G(C) predicts
the presence of internal hydrophobic domains, regions that are
characteristic of fusion proteins (Whitfield et al., 2005). The
presence of the membrane glycoproteins is essential for the
virus’s ability to replicate alternately in its plant host and its thrips
vector (Wijkamp, 1996). Evidence for the involvement of the
glycoproteins in thrips transmission is provided by the interaction
of the glycoproteins with the proteins of the thrips vector (Bandla
et al., 1998), the loss of thrips transmissibility of envelopedeficient mutants (Resende et al., 1991) and the presence of
a sequence motif that is characteristic for cellular attachment
Table 1. Genome organization of Tomato spotted wilt virus
Type (RNA) Orientation
Small (S)
Ambisense
Medium (M) Ambisense
Large (L)
Negative
Size (kb) Gene / Products
3.0
NSs / Non-structural protein
4.8
~ 9.0
N / Nucleocapsid protein
NSm / Movement protein
Function
First RNA silencing
suppressor
Protective Envelope
Cell-to-cell Movement
References
Takeda et al., 2002; Bucher et al., 2003;
Schnettler et al., 2010
de Haan et al., 1990
Storms et al., 1995; Silva et al., 2001
G1, G2 / Glycoproteins
Vector transmission
RNA dependent RNA
Polymerase
Replication
Griffiths et al., 1992; Petterson, 1991;
Stephens and Compans, 1988
Adkins et al.,1995; Chapman et al., 2003;
Van Poelwijk, 1996
74
Molecular biology of Tomato spotted wilt virus: An update
domains (Kormelink et al., 1992a). TSWV glycoproteins
were also reported to induce the formation of endoplasmic
reticulum and Golgi-derived pleomorphic membrane structures
in plant cells (Ribeiro et al., 2008). Interactions between TSWV
glycoproteins and nucleocapsid (N) proteins were studied using
fluorescence resonance energy transfer (FRET) and fluorescence
lifetime imaging microscopy (FLIM) techniques. Interaction was
demonstrated between G(C) and N and not in G (N) and N using
both the techniques (Snippe et al., 2007). Recently by using
FLIM technique, it has been studied that nucleocapsid protein
interacts with both viral glycoproteins (Riberio et al., 2009).
Glycoproteins G(N) and G(C) were examined for their lectin
binding affinity (mannose binding lectins, N-Acetyllactoseamine
lectins and fucose binding lectins) and their sensitivities to
glycosidases to know the nature of present oligosaccharides
residues on them. Result showed that G(C) has strong binding
to three lectin molecules whereas G(N) has lesser affinity to
mannose lectin but not to other two. After treatment with two
glycosidases (endoglycosidase H and peptide:N-glycosidase F
(PNGase F), there is significant decrease in the binding of G(C)
but no such effect was observed on G(N). Due to difference in
binding properties of two glycoproteins it has been suggested
that glycoprotein G(C) is heavily glycosylated as compare to
G(N). There is no evidence observed for the presence of O-linked
oligosaccharides on G (N) or G(C) (Naidu et al., 2004).
During viral infection of a plant cell, the two glycoproteins
eventually accumulate in the Golgi complex. So, site of TSWV
particle morphogenesis was determined to be the Golgi system of
host plant (Kikkert et al., 1999). Golgi stacks containing the two
glycoproteins then wrap around viral ribonucleoprotein (RNP)
complexes consisting of viral RNA, associated with nucleocapsid
(N) protein and the putative viral RNA-dependent RNA polymerase
(RdRp) to form doubly enveloped virus particles. These are thought
to fuse with each other and with ER-derived membranes, resulting
in the formation of large intracellular vesicles containing singly
enveloped virus particles (Kikkert et al., 1999).
The formation of the enveloped virus particles is strongly
regulated by the viral glycoproteins. They generally accumulate
independently at a particular cellular membrane by targeted
transport through the secretary pathway, to facilitate the interaction
with the viral nucleocapsids and the initiation of budding (Stephens
and Compans, 1988; Petterson, 1991; Griffiths et al., 1992).
S RNA: The S segment is approximately 3 kb and contains two
ORFs in ambisense orientation separated by a large intergenic
region. The ORF nearer the 5′ end of the RNA, codes for a
nonstructural protein in the viral sense designated as NSs (54
kDa). The role of the NSs protein has long been enigmatic, but
recently NSs was shown to be a suppressor of gene silencing
required to protect the virus against the plant’s anti-viral response
of post-transcriptional gene silencing (PTGS) and also affects
symptom expression in TSWV-infected plants (Takeda et al.,
2002; Bucher et al., 2003). TSWV NSs is the first RNA silencing
suppressor identified in negative-strand RNA viruses. This protein
suppressed sense transgene- induced PTGS but did not suppress
inverted repeat transgene- induced PTGS (Takeda et al., 2002).
Recently, biochemical analysis of NSs proteins from different
tospoviruses using purified NSs or NSs containing cell extracts
showed that NSs proteins have affinity to small dsRNA molecules
i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA).
The NSs protein of TSWV was shown to be capable of inhibiting
Dicer- mediated cleavage of long ds RNA in vitro. In addition, it
suppressed the accumulation of green fluorescent protein (GFP)specific siRNAs during coinfiltration with an inverted-repeatGFP RNA construct in N. benthamiana (Schnettler et al., 2010).
The ORF near the 3′ end is in viral complementary (vc) sense
and encodes for the nucleocapsid protein (N) (29 kDa) which
encapsidates the viral RNAs within the viral envelope (de Haan
et al., 1990). The nucleocapsid protein (N) contributes to the viral
replication cycle in a structural and, perhaps, regulatory manner
by participating in the complex interactions among the RNP
components leading to the initiation of viral RNA transcription
and replication. Consistent with its role in fulfilling this putative
function, the N protein has been shown to form dimers in the
absence of RNA (Uhrig et al., 1999; Kainz et al., 2004) and
to cooperatively bind ssRNA but not dsRNA (Richmond et
al., 1998). So, each RNA segment of tospovirus is associated
with nucleocapsid (N) proteins (29kDa) and a few copies of the
large (L) protein to form pseudo-circular nucleocapsid (NC)
structures or ribonucleic protein particles (RNPs) (Kormelink
et al., 1992a; Peters, 2003). These particles result from the
homopolymerization of the N protein, and are highly stable in
plant cells and can be easily purified from TSWV infected plant
cells by ultracentrifugation (de Aliva et al., 1990). On the basis
of these two properties of N protein (homopolymerization and
high stability), a gene fusion approach was explored to increase
the stability of foreign proteins produced in plants, thereby
offering a possible purification alternative for a target protein as
a gene fusion. These results show that the homopolymerization
properties of the N protein can be used as a fast and simple
way to purify large amounts of proteins from plants (Lacorte
et al., 2007). By using FRET and FLIM techniques homotypic
interactions of nucleocapsid proteins were studied (Snippe et al.,
2005). Mutated forms of N protein serve as potent dominantnegative inhibitors of virus replication (Rudolph et al., 2003).
The complete nucleotide sequence of the TSWV is now available,
allowing the precise comparison with the other animal infecting
members of the Bunyaviridae family and with other families of
plant infecting viruses (Table 2). The negative polarity of L RNA
is 8897 nucleotide long whereas ambisense M and S RNAs are
approximately 4821 and 2916 nucleotides long, respectively (de
Hann et al.,1990; 1991; Kormelink et al., 1992a).
Intergenic region (IGR): The oppositely located ORFs on the
ambisense S and M RNA segments are separated by intergenic
regions (IGRs) of several hundred nucleotides and are regarded
as the most hyper variable regions of the genome. Analysis of the
intergenic region (IGR) of S and M RNAs of tospoviruses (Family
Bunyaviridae) indicated their heterogeneity both in length and
sequence. Both IGRs contain a long stretch of mainly A residues
followed by a long stretch of mainly U residues, and are predicted
to form large stable hairpin structures (~120 bp for the S RNA,
~75 bp for the M RNA;) (de Haan et al., 1990; Kormelink et
al., 1992a). In addition, a sequence (CCAAUUUGG for S and
GCAAACUUUGG for M) that is conserved between different
tospoviruses is located near the top of these intergenic hairpins
(Maiss et al., 1991; de Haan et al., 1992; Kormelink et al., 1992a).
More specifically the 5′ and 3′ends of the IGRs are conserved,
separated by variable sequences, deletions and insertions that
appear as gaps in alignments (Bhatt et al., 1999; Heinze et al.,
2001). From the estimated sizes of the mRNAs, transcription
Molecular biology of Tomato spotted wilt virus: An update
Table 2. Complete sequence information of tripartite genome of
TSWV
Genome Strain
Nucleo- References
Database
segment No
tides (bp)
Accession No
L
8897 bp De Haan et al.,1991
NC_002052
8917 bp Unpublished*
AB190813
YN
8910bp Hu et al., 2011
JF 960237
NJ-JN 8913bp Lee et al., 2011
HM581934
CG-1
8917bp Unpublished*
JN664254
M
Rib1TL1 4786 bp Lopez et al., 2011
HM015524
Gr5TL1 4791 bp Lopez et al., 2011
HM015523
ViTL3 4787 bp Lopez et al., 2011
HM015522
NC-7
4774 bp Tsompana et al., 2005 AY744490
NC-6
4773 bp Tsompana et al., 2005 AY744489
NC-5
4787bp Tsompana et al., 2005 AY744488
NC-4
4773 bp Tsompana et al., 2005 AY744487
CA-7
4766 bp Tsompana et al., 2005 AY744485
4821 bp Kormelink et al., 1992a S48091
4821 bp Kormelink et al., 1992a NC_002050
M
4763 bp Naidu et al., 2008
AY870390
T
4774 bp Naidu et al., 2008
AY870389
4768 bp Unpublished*
AB190818
* Taken from www.ncbi.nlm.nih.gov (nucleotide database)
is thought to terminate somewhere in the IR (de Haan et al.,
1990; Kormelink et al., 1992a), and it has been suggested that
the hairpin structure or the conserved sequence motif may be
involved in transcription termination (van Knnipenberg et al.,
2005).
There is the first report on detailed sequence analysis of IGRs of
S and M RNAs of known tospoviruses (Pappu et al., 2000). In
general, IGRs of M RNA were shorter in length compared to the
IGRs of their respective S RNA species. Per cent identity among
the S RNA IGR sequences of distinct tospovirus species varied
from 42 to 57%, whereas it was 79 to 99% among isolates of the
same species. Similarly, when IGRs of M RNAs were compared,
there was higher sequence identity among isolates of the same
tospovirus species (84 to 98%) than among distinct tospovirus
species (46 to 59%). Per cent nucleotide identities and maximum
likelihood trees of IGR sequences of S and M RNAs indicated
that their sequence divergence is similar to that of nucleocapsid
gene at inter and intra-species levels (Pappu et al., 2000). The
intergenic region (IGR) of the medium (M) RNA of TSWV
isolates naturally infecting peanut (groundnut), pepper, potato,
stokesia, tobacco and watermelon in Georgia (GA) and a peanut
isolate from Florida (FL) was analysed. The IGR sequences were
compared with one another and with respective M RNA IGRs
of TSWV isolates from Brazil and Japan and other tospoviruses.
The length of M IGR of GA and FL isolates varied from 271 to
277 nucleotides. IGR sequences were more conserved (95-100%)
among the populations of TSWV from GA and FL, than when
compared with those of TSWV isolates from other countries
(83-94%). Cluster analysis of the IGR sequences showed that all
GA and FL isolates are closely clustered and are distinct from
the TSWV isolates from other countries as well as from other
tospoviruses (Bhatt et al., 1999).
Transcription: TSWV initiates transcription of their genome
by a mechanism called cap snatching. All TSWV genes are
expressed by the synthesis of mRNAs that can be discriminated
from the (anti) genomic RNA strands by the presence of non-
75
viral leader sequences (Kormelink et al., 1992b and 1992c).
For these leader sequences, TSWV has evolved a cap stealing
mechanism known as cap snatching. During this process the
viral RNA dependent RNA polymerase (RdRp), encompassing
an endonuclease activity, cleaves a host mRNA at a position 1020 nucleotides from the capped 5′ end of host mRNA. The short
capped fragments act as primers for viral mRNA transcription
(Kormelink et al., 1992c; Van Poelwijk et al., 1996; Duijsings
et al., 2001). This mechanism is used by all segmented negative
strand RNA viruses to initiate transcription of their genome
and was first described for Influenza virus (Plotch et al., 1981;
Nguyen and Haenni, 2003). This mechanism was investigated
for TSWV by extensive inplanta studies (Duijsings et al., 1999;
2001), resulting in the model for transcription initiation. It has
been demonstrated that Alfalfa mosaic virus (AMV) RNAs can
be utilized by TSWV as cap donors during a mixed infection of N.
benthamiana (Duijsings et al., 1999). Furthermore, it was shown
that suitable cap donors require a single base complementarity to the
ultimate or penultimate residue of the TSWV template (Duijsings
et al., 2001). It has been demonstrated that in vitro ongoing
transcription of TSWV requires the presence of reticulocyte lysate.
This dependence was further investigated by testing the occurrence
of transcription in the presence of two translation inhibitors: edeine,
an inhibitor that still allows scanning of nascent mRNAs by the 40S
ribosomal subunit, and cycloheximide, an inhibitor that completely
blocks translation including ribosome scanning. Neither of these
inhibitors blocked TSWV transcription initiation or elongation
in vitro, as demonstrated by de novo-synthesized viral mRNAs
with globin mRNA-derived leader sequences, suggesting that
TSWV transcription in vitro requires the presence of (a component
within) reticulocyte lysate, rather than a viral protein resulting from
translation (van Knippenberg et al., 2004).
Thrips -vector of TSWV: Cell walls present in plants are major
barrier to viral infection. Some plant viruses depend upon vector
(vehicles) for their movement from infected host plants to healthy
ones. Insects (thrips) play a very important role as vector in virus
transmission. Thrips are important members of the ecosystems as
herbivores and predators. Thrips are minute slender bodied insects
that have ability to transmit plant viruses. They are categorized
as important agricultural pest. Thrips belong to the insect order
Thysanoptera and the main TSWV vector species all belong to
family Thripidae. Thrips feed on plant tissue with piercing and
sucking mouth parts. Thrips acquire TSWV in two larval stages
and it is only when larvae feed on infected plant host. Once thrips
acquire the virus they remain viruliferous (infected with virus)
throughout their life span. Virus acquired by the larvae renders
the thrips infectious, and transmission of the virus is mainly
ascribed to adults (Sakimura, 1962). It has been studied that
TSWV infection alters the feeding behavior of its insect vector.
Data reveals that viruliferous males are good feeder as compare to
non- viruliferous males and for females no change was observed
for their behavior (Stafford et al., 2010). Wijkamp et al. (1993)
showed that larvae of Frankliniella occidentalis also transmit the
virus efficiently. The virus upon acquisition was shown to move
through the midgut and subsequently reaches the salivary glands.
It is hypothesized that the close proximity of midgut and salivary
glands in the thrip’s larval stage facilitates the virus movement
whereas the virus fails to do so as the thrips reaches adult stage.
This may explain the inability of adult thrips to transmit the virus
if the virus is acquired for the first time in its adult life (Filho et
76
Molecular biology of Tomato spotted wilt virus: An update
al., 2004). The specificity of TSWV and thrips vectors may be
due to the presence of a receptor in the vector species which may
be absent in non-vector species. F. occidentalis and Thrips tabaci
have been found to be vectors of at least four important plant
virus groups including the bunyaviruses (Ullman et al., 1997).
Earlier, eight species of thrips were reported to transmit TSWV
(Wijkamp et al., 1995). F. occidentalis Pergabnde (the western
flower thrips), T. tabaci Lindeman (the onion thrips), T. palmi
Karny (melon thrips), T. setosus Moulton, F. schultzei Trybom
(the common blossom or cotton bud thrips), F. intonsa Trybom
and F. fusca Hinds (the tobacco thrips) were reported to be vector
of TSWV (Wijkamp et al., 1995; Ullman et al., 1997). Webb et
al. (1997) also reported F. bispinosa Morgan (Florida flower
thrips) as a vector of TSWV. F. tenuicornis Uzel (European
grass thrips) and Scirtothrips dorsalis Hood (the chilli thrips) had
been previously reported to be vector of TSWV, but experimental
verification had not been done for all species as has been done for
F. occidentalis and T. tabaci (Ullman et al., 1997). F. occidentalis
and T. tabaci are common and important vectors of multiple plant
viruses in many regions of the world (Ullman et al., 1997).
Molecular mechanism of transmission: Insect vectors play a
key role in dissemination of viruses that cause important diseases
in humans, animals and plants (Ullman et al., 2005). With the
discovery that TSWV multiplies in insect vectors, the complex
nature of the interplay between thrips, tospoviruses and their
plant hosts was first recognized by Ullman et al. (1993) and
Wijkamp et al. (1993). TSWV is able to infect both its botanical
hosts and its insect vector (thrips). It has been demonstrated by
using the approach of Reddy and Black (1966) that tospovirus
multiply in their insect vector (F. occidentalis). The evidence
that genetic determinants for TSWV transmissibility reside on
middle RNA which encode viral glycoproteins came by creating
reassortants by coinoculating plants with thrips-transmissible
isolate (TSWV-RG2) and a thrips-nontransmissible TSWV isolate
(TSWV-D) (Sin et al., 2005). Insect inoculation of tospoviruses
into their plant hosts cannot occur without viral passage across
at least three insect organs (the midgut, visceral muscle cells and
salivary glands) that include six membrane barriers (Whitfield
et al., 2005). Replication of the TSWV in midgut, its movement
from midgut to visceral muscles and then the salivary glands are
crucial factor determining the vector competency (Nagata et al.,
2002). Several lines of experiments demonstrated that TSWV GPs
bind to the insect midgut during TSWV acquisition by thrips and
plays a critical role in TSWV transmission while TSWV G(N)
binds the insect vector midgut and inhibits TSWV acquisition
(Ullman et al., 2005). Whitfield et al. (2008) also reported that
soluble form of the envelope glycoprotein GN (GN-S) specifically
bound thrips midguts and reduced the amount of detectable virus
inside midgut tissues.
An increase in the concentration of two TSWV encoded proteins
(N & NSs), by S RNA firmly demonstrate replication of TSWV
in its vector. The accumulation of N protein is indicative of the
production of virus particles, but the accumulation of the NSs
protein, which has not been found in virus particles (Kormelink
et al., 1991) can only occur after transcription of its mRNA from
the complementary viral RNA strand which is formed during the
replication of viral RNA. Hence, the presence and increase of this
protein give conclusive proof that TSWV replicates in its vector
(F. occidentalis) (Wijkamp et al., 1993).
Screening of a cDNA library of F. occidentalis using fragments
of TSWV RdRp, a F. occidentalis putative transcription factor
(FoTF) was identified that binds to TSWV RdRp, which was
shown to bind to TSWV RNA and enhance TSWV replication
(in vitro). Mammalian cells expressing this putative transcription
factor supported TSWV replication. So, this factor which supports
TSWV replication in vivo and in vitro could be used to compare
molecular defense mechanisms in plant, insect and mammalian
cell lines against the same pathogen for better understanding of
evolutionary studies (Medeiros et al., 2005).
Molecular detection and characterization: TSWV has certain
unique biological properties that are useful for diagnosis. A
turning point in TSWV detection and diagnosis came with the
production of high quality polyclonal antisera and development
of an enzyme-linked immunosorbent assay (ELISA) (Gonsalves
and Trujillo, 1986). High quality specific polyclonal antisera has
also been developed for virus detection using in vitro expressed
coat protein of different viruses like Carnation etched ring virus
(CERV) and Prunus necrotic ring spot virus (PNRSV) (Raikhy
et al., 2007; Kulshrestha et al., 2009). Adam et al. (1995) have
described an assay which could detect tospoviruses generally,
based on antibodies to the G proteins of the virus. Dot-blot
immunoassay and tissue-print immunoassay has also been
used for specific detection of TSWV (Hsu & Lawson, 1991;
Louro, 1995). TSWV detection by molecular methods has been
developed using cDNA probes (Ronco et al., 1989; Rice et al.,
1990) and riboprobes (Huguenot et al., 1990), both of which
have proved useful for the sanitary certification of plant material
(Saldarelli et al., 1996). Several PCR-based methods have also
been developed for the specific detection of TSWV. The first
PCR-based assay was developed by Mumford et al. (1994).
Immunocapture PCR and RT-PCR were developed by Nolasco
et al. (1993) and Weekes et al. (1996), respectively. A very
sensitive protocol for the detection and quantification of TSWV
is the real-time RT-PCR assay which is based on TaqManTM
chemistry, on both “leaf soak” and total RNA extracts from
infected plants (Roberts et al., 2000). A comparison between
ELISA and RT-PCR assays was done to detect TSWV in fieldgrown chrysanthemum (Matsuura et al., 2002; 2004) and recently
for peanut also (Dang et al., 2009). Similarly potential of RT- PCR
was evaluated by comparing its sensitivity with DAS-ELISA
for the detection of TSWV among 22 Australian plant species.
DAS ELISA was found to be less sensitive as compare to RTPCR method (Dietzgen et al., 2005). One-step multiplex reverse
transcription-polymerase chain reaction (multi-PCR) (modified)
was also utilized for simultaneous identification of five tospovirus
species (Kuwbara et al., 2010). For the detection of TSWV in
individual thrips, a sensitive and robust real time fluorescent
(RT-PCR Taqman) technique was developed (Boonham et al.,
2002). Similarly, by using RT-PCR technique TSWV from a
single infected thrip has been reported by Mason et al. (2003).
A quantitative real-time reverse transcription-polymerase chain
reaction (RT-qPCR) procedure using a general primer set and
three TaqMan(®)MGB probes was developed for general and
genotype-specific detection and quantization of the genomic M
segment of TSWV (Debreczeni et al., 2011).
Similarly PCR based detection methods were also developed
for the detection of other economically important viruses like
PNRSV in pelargonium (Kulshrestha et al., 2005c), Tuberose
Molecular biology of Tomato spotted wilt virus: An update
mild mottle virus in Tuberose (Kulshrestha et al., 2005b).
Nematode transmitted viruses like Arabis mosaic virus (ArMV)
and Strawberry latent ringspot virus (SLRSV) has also been
detected in vector nematodes by RT-PCR (Kulshrestha et al.,
2005a; Adekunle et al., 2005). In Korea, TSWV is characterized
from paparika (Capsicum annuum var. grossum). TSWV-KP
nucleocapsid (N) protein of the purified virion migrated as a single
band with molecular weight of about 29 kDa in SDS-PAGE. The N
gene of TSWV-KP showed 96.5-97.2 % and 97.7-98.5 % identities
to the three different TSWV isolates of Genbank Database at the
nucleotide and amino acid, respectively (Kim et al.,2004). The
complete cDNA genomic libraries were prepared from complete
genomic RNA of Bulgerian L3 isolate of TSWV. S RNA specific
clones were selected which covered approximately 2.8kb which
is 95 % of this RNA and S RNA to be ambisense in nature (Maiss
et al., 1991). In Israel, virus identity was characterized by host
range, serology and electron microscopy. Serological reaction
with the isolates, found in Israel, using antisera from different
sources as well as the sequence analysis of the nucleocapsid gene,
demonstrated that the Israeli isolates of TSWV are a member of
tospovirus serogroup I, type I (BR-01 strain) (Antignus et al.,
1997). There was a first report of molecular characterization of
TSWV on pepper (Capsicum sp.) in South Africa (SA) by taking
six isolates from different geographical locations of the country.
The identity of this virus was confirmed by using ELISA, electron
microscopy and protein analysis. Phylogenetic analysis based on
multiple alignments of N gene sequences revealed the branching
of TSWV in two distinct clusters designated as the American and
European groups. TSWV from SA showed high sequence similarity
with TSWV isolates from Europe. Among themselves, the N
gene of six isolates revealed high similarity (> 90 %) (Sivprasad
and Gubba, 2008). Genetic variability was analyzed for TSWV
from different crop species in Australia. It was found that genetic
variability was small among Australian isolates (Dietzgen et al.,
2005). In Jordan, TSWV infection was recorded for the first time
and sequence analysis of isolates from Jordan shared nucleotide
similarities with isolates from different countries (Anfoka et al.,
2006). In China, complete nucleotide sequence of TSWV isolate
was determined and phylogenetic relationship of the Chinese isolate
was also analyzed from Brazilian and Korean isolates on the basis
of nucleotide sequences of the glycoprotein and nucleocapsid genes
(Hu et al., 2011).
The review specifically focuses on the molecular biology of
Tomato spotted wilt virus (TSWV). Earlier reviews which
have been published focuses on Tospovirus genus (Adkins,
2000; Elliot, 1990; Goldbach and Peters, 1996; Pappu, 2008;
Tsompana and Moyer, 2008). Important focus of this review is
on the function of different TSWV genome encoded proteins
like N and NSs by small RNA. N proteins functions as
protective envelope and NSs protein (non structural protein)
has been recently identified as first RNA silencing suppressor
which is responsible for inhibition of dicer mediated cleavage
(Bucher et al., 2003; Schnettler et al., 2010 and Takeda et al.,
2002). Medium RNA encodes NSm which helps in cell to cell
movement; G1/G2 glycoproteins help in vector transmission.
Similarly L RNA segment of TSWV encodes for RNA dependant
RNA polymerase which help in replication. Out of these
proteins, amino acid sequences of N protein were used for the
identification of new tospovirus species, though exceptions
77
remain (Hassani-Mehraban et al., 2007). This review also
discusses possible mechanism of TSWV transmission by thrips
(F. occidentalis) along with different known factors from both
virus and vector side which plays critical role in virus acquisition
by thrips and its subsequent transfer.
Till date there is no report of TSWV infection from India, but it has
been reported form several parts of the world. During our survey in
different districts of Himachal Pradesh, TSWV like symptoms were
observed on capsicum and tomato, so there is an urgent need for an
extensive survey to rule out the possibility of TSWV infection in
India. There is also an urgent need to have strict quarintine norms
to restrict the entry of TSWV from other parts of the world.
Acknowledgements
The authors thank Prof. P. K. Khosla, Hon’ble Vice-Chancellor,
Shoolini University of Biotechnology and Management Sciences,
Solan and Foundation for Life Sciences and Business Management
(FLSBM), Solan for providing financial support and necessary
facilities. We are also indebted to Prof. D.R. Sharma for critical
review of the paper and helpful comments.
References
Adkins, S. 2000. Tomato spotted wilt virus--positive steps towards
negative success. Molecular Plant Pathol., 1: 151- 157.
Adkins, S., R. Quadt, T.J. Choi, P. Ahlquist and T. German, 1995. An
RNA-dependent RNA polymerase activity associated with virions of
Tomato spotted wilt virus, a plant- and insect-infecting bunyavirus.
Virol., 207: 308-311.
Adekunle, O.K., S. Kulshrestha, R. Prasad, V. Hallan, G. Raikhy, N.
Verma, R. Ram, S. Kumar and A.A. Zaidi, 2005. Plant parasitic and
vector nematodes associated with Asiatic and Oriental hybrid lilies.
Bioresour. Technol., 97: 364-371.
Adam, G., P. Roggero, F. Malavasi, R.G. Milne and G. Papa, 1995.
Approach to a gene tospovirus assay using antibodies to purified
Tomato spotted wilt tospovirus G proteins. Bulletin OEPP/EPPO
Bulletin., 25: 247-257.
Antignus, Y., M. Lapidot, N. Ganaim, J. Cohen, O. Lachman,
M. Pearlsman, B. Raccah and A. Gera, 1997. Biological and
molecular characterization of Tomato spotted wilt virus in Israel.
Phytoparasitica, 25: 319-330.
Anfoka, G.H., M. Abhary and M.R. Stevens, 2006. Occurence of Tomato
spotted wilt virus (TSWV) in Jordan. EPPO Bulletin., 36: 517-522.
Bandla, M.D., L.R. Campbell, D.E. Ullman and J.L. Sherwood,1998.
Interaction of Tomato spotted wilt Tospovirus (TSWV) glycoproteins
with a thrips midgut protein, a potential cellular receptor for TSWV.
Phytopathol., 88: 98-104.
Best, R.J. 1968. Tomato spotted wilt virus. Adv.Virus Res., 13: 65-146.
Best, R.J. and B.A. Palk, 1964. Electron microscopy of strain E of Tomato
spotted wilt virus and comments on its probable biosynthesis. Virol.,
23: 445-460.
Bhatt, A.I., S.S. Pappu, H.R. Pappu, C.M. Deom and A.K. Culbreath,
1999. Analysis of the intergenic region of Tomato spotted wilt
Tospovirus medium RNA segment. Virus Res., 61: 161-170.
Boonham, N., P. Smith, K. Walsh, J. Tame, J. Morris, N. Spence, J.
Bennison and I. Barker, 2002. The detection of Tomato spotted wilt
virus (TSWV) in individual thrips using real time fluorescent RTPCR (TaqMan). J. Virol. Methods, 101: 37-48.
Brittlebank, C.C., 1919. Tomato diseases. J. Agr. Victoria., 17: 213-23.
Bucher, E., T. Sijen, P. De Haan, R. Goldbach, M. Prins, 2003. Negativestrand Tospoviruses and Tenuiviruses carry a gene for a suppressor
of gene silencing at analogous genomic positions. J. Virol., 77:
1329-1336.
78
Molecular biology of Tomato spotted wilt virus: An update
Chapman, E.J., P. Hilson, T.L. German, 2003. Association of L protein
and in vitro Tomato spotted wilt virus RNA-dependent RNA
polymerase activity. Intervirol., 46: 177-181.
Dang, P.M., D.L. Rowland and W.H. Faircloth, 2009. Comparison of
ELISA and RT-PCR assays for the detection of Tomato spotted wilt
virus in peanut. Peanut Sci., 36: 133-137.
de Avila, A., C.C. Huguenot, R. De. O. Resende, E. Kitajima, W.R.W.
Goldbach and D. Peters, 1990. Serological differentiation of 20 isolates
of Tomato spotted wilt virus. J. Gen. Virol., 71: 2801-2807.
de Aliva, A.C. 1992. Diversity of Tospoviruses, p. 136. PhD Thesis,
Wageningen Agricultural University, Wageningen, Netherlands.
Debreczeni, D.E., S. Ruiz-Ruiz, J. Aramburu, C. López , B. Belliure, L.
Galipienso, S. Soler and L. Rubio, 2011. Detection, discrimination
and absolute quantification of Tomato spotted wilt virus isolates
using real time RT-PCR with TaqMan(®)MGB probes. J. Virol.
Methods, 176: 32-37.
de Haan, P., L. Wagemakers, D. Peters and R. Goldbach, 1990. The
S RNA segment of Tomato spotted wilt virus has an ambisense
character. J. Gen. Virol., 71: 1001-1007.
de Haan, P., J.J.L. Gielen, M. Prins, I.G. Wijkamp, A. Van Schepen,
D. Peters, M.Q.J.M. Van Grinsven and R. Goldbach, 1992.
Characterization of RNA-mediated resistance to Tomato spotted wilt
virus in transgenic tobacco plants. BioTechnol., 10: 1132-1137.
de Haan, P., R. Kormelink, R. De. O. Resende, F. van Poelwijk, D. Peters
and R. Goldbach, 1991. Tomato spotted wilt virus L RNA encodes a
putative RNA polymerase. J. Gen. Virol., 72: 2207-2216.
de Haan, P. L. Wagemakers, D. Peters and R. Goldbach, 1989. Molecular
cloning and terminal sequence determination of the S and M RNAs
of Tomato spotted wilt virus. J. Gen. Virol., 70: 3469-3473.
Dietzgen, R.G., J. Twin, J. Talty, S. Selladurai, M.L. Carroll, B.A.
Coutts, D.I. Berryman and R.A.C. Jones, 2005. Genetic variability
of Tomato spotted wilt virus in Australia and validation of real time
RT-PCR for its detection in single and bulked leaf samples. Ann.
Appl. Biol., 146: 517-530.
Duijsings, D., R. Kormelink and R. Goldbach, 1999. Alfalfa mosaic
virus RNAs serve as cap donors for Tomato spotted wilt virus
transcription during co infection of Nicotiana benthamiana. J. Virol.,
73: 5172- 5175.
Duijsings, D., R. Kormelink and R. Goldbach, 2001. In vivo analysis of
the TSWV cap-snatching mechanism: Single base complementarity
and primer length requirements. EMBO J., 20: 1.
Elliot, R.M. 1990. Molecular biology of the Bunyaviridae. J. Gen. Virol.,
71: 501-522.
Elliott, R.M., C. Bouloy, C.H. Calsher, R. Goldbach, J.T. Moyer, S.T.
Nichol, R. Petterson, A. Plyusnin and C.S. Schmaljohn, 2000. Family
Bunyaviridae. In: Virus Taxonomy VIIth Report of the International
Committee on Taxonomy of Viruses. M.H.V. Van Regenmortel, C.M.
Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J.
Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle, R.B. Wickner
(eds.). Academic Press, USA. pp. 599-621.
Filho Assis de, F.M., C.M. Deom and J.L. Sherwood, 2004. Acquisition
of Tomato spotted wilt virus by adults of two thrips species.
Phytopathol., 94(4): 333-336.
Geerts- Dimitriadou, C., Y.Y. Lu, C. Geertsema, R. Goldbach and
R. Kormelink, 2012. Analysis of the Tomato spotted wilt virus
ambisense S RNA-encoded hairpin structure in translation. PLoS
One, 7(2):310-13.
Gera, A., J. Kritzman, J. Cohen, B. Racca and Y. Antignus, 2000.
Tospoviruses infecting vegetable crops in Israel. EPPO Bulletin,
30: 289-292.
German, T.L., D.E. Ullman and J.W. Moyer, 1992. Tospoviruses:
diagnosis, molecular biology, phylogeny and vector relationships.
Annu. Rev. Phytopathol., 30: 315-348.
Griffiths, G. and P. Rottier, 1992. Cell biology of viruses that assemble
along the biosynthetic pathway. Semin. Cell Biol., 3: 367-381.
Goldbach, R and D. Peters, 1996. Molecular and biological aspects of
tospoviruses. In: The Bunyaviridae. R.M. Elliott (ed.). Plenum Press,
New York, USA.
Gonsalves, D. and E.E. Trujillo, 1986. Tomato spotted wilt virus in papaya
and detection of the virus by ELISA. Plant Dis., 70: 501-506.
Hassani-Mehraban, A., J. Saaijer, D. Peters, R. Goldbach and R.
Kormelink, 2007. Molecular and biological comparison of two
Tomato yellow ring virus (TYRV) isolates: challenging the
Tospovirus species concept. Arch. Virol., 152: 85- 96.
Heinze, C., B. Letschert, D. Hristova, M. Yankulova, O. Kauadjouor, P.
Willingmann, A. Atanassov and G. Adam, 2001. Variability of the
N-protein and the intergenic region of the S RNA of Tomato spotted
wilt Tospovirus (TSWV). New Microbiol., 24: 175-187.
Hu, Z.Z., Z.K. Feng, Z.J. Zhang, Y.B. Liu and X.R.Tao, 2011. Complete
genome sequence of Tomato spotted wilt virus isolate from China
and comparison to other TSWV isolates of different geographical
region. Arch. Virol., 156: 1905-1908.
Huguenot, C., Van. G. Dobbelsteen, P. Den Haan, C.A.M. De Wagemakers,
G.A. Drost, A.D.M.E. Osterhausand and D. Peters, 1990. Detection
of Tomato spotted wilt virus using monoclonalantibodies and
riboprobes. Arch. Virol., 110: 47-62.
Hsu, H.T. and R.H. Lawson, 1991. Direct tissue blotting for detection of
Tomato spotted wilt virus in Impatiens. Plant Dis., 75: 292-295.
Ie, T.S. 1964. An electron microscopestudy of Tomato spotted wilt virus
in the plant cell. Neth. J. Plant Pathol., 70: 114-115.
Kainz, M., P. Hilson, L. Sweeney, E. DeRose and T.L. German, 2004.
Interaction between Tomato spotted wilt virus N protein monomers
involves non-electrostatic forces governed by multiple distinct
regions of the primary structure. Phytopathol., 94: 759- 65.
Kikkert, M., J. Van Lent, M. Storms, P. Bodegom, R. Kormelink and R.
Goldbach, 1999. Tomato spotted wilt virus particle morphogenesis
in plant cells. J. Virol., 73: 2288-97.
Kim, J.H., G.S. Choi, J.S. Kim and J.K. Choi, 2004. Characterization
of Tomato spotted wilt virus from paprika in Korea. Plant Pathol.
J., 20: 297-301.
Kitajima, E.W. 1965. Electron microscopy of vira- cabeca virus (Brazilian
Tomato spotted wilt virus) within the host cell. Virol., 26: 88-99.
Kormelink, R., P. de Haan, C. Meurs, D. Peters and R. Goldbach, 1992a.
The nucleotide sequence of the M RNA segment of Tomato spotted
wilt virus, a bunyavirus with two ambisense RNA segments. J. Gen.
Virol., 73: 2795-2804.
Kormelink, R., P. de Haan, D. Peters and R. Goldbach, 1992b. Viral RNA
synthesis in Tomato spotted wilt virus-infected Nicotiana rustica
plants. J. Gen. Virol., 73: 687.
Kormelink, R., F. Van Poelwijk, D. Petersand and R. Goldbach, 1992c.
Non-viral heterogeneous sequences at the 5´ ends of Tomato spotted
wilt virus mRNAs. J.Gen. Virol., 73: 2125- 2128.
Kormelink, R., E.W. Kitajima, P. de Haan, D. Zuidema, D. Peters and
R. Goldbach, 1991. The non structural protein (NSs) encoded by the
ambisense S RNA segment of Tomato spotted wilt virus is associated
with fibrous structures in infected plant cells. Virol., 181: 459-468.
Kuwabra, K., N. Yokoi, T. Ohki and S. Tsuda, 2010. Improved multiplex
reverse transcription-polymerase chain reaction to detect and identify
five tospovirus species simultaneously. J. Gen. Plant Pathol., 76:
273-277.
Kulshrestha, S., V. Hallan, S. Raikhy and A.A. Zaidi, 2009. Potential
uses of in vitro expressed and purified recombinant Prunus
necrotic ringspot virus coat protein gene. Arch. Phytopathol. Plant
Protection., 42: 442-452.
Kulshrestha, S., V. Hallan, G. Raikhy, O.K. Adekunle, Q.M.R. Haq and
A.A. Zaidi, 2005a. Reverse transcription polymerase chain reactionbased detection of Arabis mosaic virus and Strawberry latent
ringspot virus in vector Nematodes. Curr. Sci., 89: 1759-1762.
Kulshrestha, S., A. Mehra, V. Hallan, G. Raikhy, R. Ram and A.A.
Zaidi, 2005b. Molecular evidence for the occurrence of Tuberose
mild mottle virus infecting tuberose (Polianthes tuberosa) in India.
Curr. Sci., 89: 870-872.
Kulshrestha, S., N. Verma, V. Hallan, V. Raikhy, M.K. Singh, R. Ram and
A.A. Zaidi, 2005c. Detection and identification of Prunus necrotic
ringspot virus in Pelargonium. Austral. Plant Pathol., 34: 599-601.
Molecular biology of Tomato spotted wilt virus: An update
Lacorte, C., S.G. Ribeiro, D. Lohuis, R. Goldbach and M. Prins, 2007.
The nucleoprotein of Tomato spotted wilt virus as protein tag for
easy purification and enhanced production of recombinant proteins
in plants. Prot. Expt. Purif., 55: 17-22.
Lee, J.S., W.K. Cho, M.K. Kim, H.R. Kwak, H.S. Choi and K.H. Kim,
2011. Complete genome sequences of three Tomato spotted wilt virus
isolates from tomato and pepper plants in Korea and their phylogenetic
relationship to other TSWV isolates. Arch. Virol., 156: 725-728.
Li, W., D.J. Lewandowaski, M.E. Hiff and S. Adkins, 2010. Insights
into common functional domains of tospovirus NSm proteins.
Phytopathol., 100:70.
Lopez, C., J. Aramburu, L. Galipienso, S. Soler, F. Nuez and L. Rubio,
2011. Evolutionary analysis of tomato Sw-5 resistance breaking
isolates of Tomato spotted wilt virus. J. Gen. Virol., 92: 210-215.
Louro, D. 1995. Use of tissue-print immunoassay for the practical
diagnosis of Tomato spotted wilt tospovirus. Bulletin OEPP/EPPO
Bulletin., 25: 277-281.
Mason, G., P. Roggero and L. Tavella, 2003. Detection of Tomato spotted
wilt virus in its vector Frankliniella occidentalis by reverse trascriptionpolymerase chain reaction. J. Virol. Methods, 109: 69-73.
Matsuura, S., S. Hoshino, H. Yayashi, T. Kohguchi, K. Hagiwara and
T. Omura, 2002. Effects of latent infection of stock plants and
abundance of thrips on the occurrence of Tomato spotted wilt virus
in Chrysanthemum fields. J. Gen. Plant Pathol., 68: 99-102.
Matsuura, S., S. Ishikura, N. Shigemoto, S. Kajihara and K. Hagiwara,
2004. Localization of Tomato spotted wilt virus in Chrysanthemum
stock plants and efficiency of viral transmission from infected stock
plants to cuttings. J. Phytopathol., 152: 219-223.
Martin, M.M. 1964. Purification and electron microscope studies of
Tomato spotted wilt virus (TSWV) from tomato roots. Virol., 22:
645-649.
Maiss, E., L. Ivanova, E. Breyel and G. Adam, 1991. Cloning and
sequencing of the S RNA from a Bulgarian isolate of Tomato spotted
wilt virus. J. Gen. Virol., 72: 461.
Milne, R.J. 1993. Electron microspcopy of in vitro preparations. In:
Diagnosis of Plant Virus Dis. R.E.F. Matthews (ed.). CRC Press,
USA. p. 215-251.
Medeiros, R.B., J. De. Fiueriredo, R. de O. Resende and A.C. de Aliva,
2005. Expression of a viral polymerase- bound host factor turns
human cell lines permissive to plant and insect infecting virus. Proc.
Natl. Acad. Sci. USA., 102: 1175-1180.
Moya, A., S.F. Elena, A. Bracho and R. Miralles Barrio, 2000. The
evolution of RNA viruses: a population genetics view. Proc. Natl.
Acad. Sci. USA., 97: 6967-6973.
Moyer, J.W. 1999. Tospoviruses (Bunyaviridae). In: Encycl. of Virol. A.
Granoff, R. G. Webster (eds.). Academic Press, San Diego, USA.
pp. 1803-1807.
Moyer, J.W. 2000. Tospoviruses. In: Encyclopedia of Microbiology. R.
Hull (ed.). Academic Press, London. p. 592-597.
Mumford, R.A., I. Barker and K.R. Wood, 1996. The biology of the
tospoviruses. Ann. Appl. Biol., 128: 159-183.
Mumford, R.A., I. Barker and K.R .Wood, 1994. The detection of Tomato
spotted wilt virus using the polymerase chain reaction. J. Virol.
Methods, 46: 303-311.
Nagata, T., A.K. Inoue-Nagata, J.V. Lent, R. Goldbach and D. Peters,
2002. Factors determining vector competence and specificity for
transmission of Tomato spotted wilt virus. J. Gen. Virol., 83: 66371.
Naidu, R.A., C.J. Ingle, C.M. Deom and J.L. Sherwood, 2004. The two
envelope membrane glycoproteins of Tomato spotted wilt virus
show differences in lectin-binding properties and sensitivities to
glycosidases. Virol., 319: 107-117.
Naidu, R.A., J.L. Sherwood and J.L. Deom, 2008. Characterization of a
vector-nontransmissible isolate of Tomato spotted wilt virus. Plant
Pathol., 57: 190-200.
Nguyen, M. and A.L. Haenni, 2003. Expression strategies of ambisense
viruses. Virus Res., 93: 141-150.
79
Nolasco, G., C. de Blas, V. Torres and F. Ponz, 1993. A method combining
immunocapture and PCR amplification in a micro titer plate for
the detection of a plant viruses and sub viral pathogens. J. Virol.
Methods., 45: 201-218.
Pappu, H.R. 2008. Tomato spotted wilt virus (Bunyaviridae). In: Encycl.
of Virol. B.W.J. Mahy, M.H.V. Van Regenmortel (eds.). Elsevier,
Oxford, UK. p.133-138.
Pappu, S.S., A.I. Bhat, H.R. Pappu, C.M. Deom and A.K. Culbreath,
2000. Phylogenetic studies of tospoviruses (family: Bunyaviridae)
based on intergenic region sequences of small and medium genomic
RNAs. Arch. Virol., 145: 1035-45.
Parrella, G., P. Gognalons, K. Gebre-Selassiè, C. Vovlas and G.
Marchoux, 2003. An update of the host range of Tomato spotted wilt
virus. J. Plant Pathol., 85: 227-264.
Peters, D. 2003. Tospoviruses. In: Viruses and Virus-like Diseases
of Major Crops in Developing Countries .G. Loebenstein, G.
Thottappilly (eds.). Kluwer Academic Publishers, USA. p.719742.
Peters, D., R. Goldbach, 1995. The biology of tospoviruses. In:
Pathogenesis and Host Specificity in Plant Disease. Vol. III Viruses
&Viroids. R.P. Singh, U.S. Singh, K. Kohmoto (eds.). Pergamon
Press, Oxford. p. 199-210.
Petterson, R.F. 1991. Protein localization and virus assembly at intracellular
membranes. Curr. Top. Microbiol. Immunol., 170: 67-104.
Plotch, S.J., M. Bouloy, I. Ulmanen and R.M. Krug, 1981. A unique
cap (m7GpppXm) - dependent Influenza virion endonuclease
cleaves capped RNAs to generate the primers that initiate viral RNA
transcription. Cell., 23: 847-858.
Prins, M and R. Goldbach, 1998. The emerging problem of tospovirus
infection and nonconventional methods of control. Trends Microbiol.,
6: 31-35.
Raikhy, G., V. Hallan, S. Kulshrestha and A.A. Zaidi, 2007. Polyclonal
antibodies to the coat protein of Carnation etched ring virus expressed
in bacterial system: Production & use in immunodiagnosis. J.
Phytopathol., 155: 616-622.
Resende, R.O., P. de Haan, A.C. de Avila, E.W. Kitajima, R. Kormelink,
R. Goldbach and D. Peters, 1991. Generation of envelope and
defective interfering RNA mutants of Tomato spotted wilt virus by
mechanical passage. J. Gen. Virol., 72: 2375-2383.
Reddy, D.V.R. and L.M. Black, 1966. Production of Wound tumor virus
and wound tumor soluble antigen in the insect vector. Virol., 30:
551-561.
Ribeiro, D., O. Foresti, J. Denecke, J. Wellink, R. Goldbach and R.J.M.
Kormelink, 2008. Tomato spotted wilt virus glycoproteins induce the
formation of endoplasmic reticulum- and Golgi-derived pleomorphic
membrane structures in plant cells. J. Gen. Virol., 89: 1811-1818.
Ribeiro, D., J.W. Brost, R. Goldbach and R.J.M. Kormelink, 2009.
Tomato spotted wilt virus nucleocapsid protein interacts with both
viral glycoproteins Gn and Gc in Planta. Virol., 383.
Rice, D., T.L. German, R.F.L. Mau and F.M. Fujimoto, 1990. Dot blot
detection of Tomato spotted wilt virus RNA in plant and thrips tissues
by cDNA clones. Plant Dis., 74: 274-276.
Richmond, K.E., K. Chenault, J.L. Sherwood and T.L. German, 1998.
Characterization of the nucleic acid binding properties of Tomato
spotted wilt virus nucleocapsid protein. Virol., 248: 6-11.
Roberts, C.A., R.G. Dietzgen, L.A. Heelan and D.Maclean, 2000.Realtime RT-PCR fluorescent detection of Tomato spotted wilt virus. J.
Virol. Methods, 88: 1-8.
Ronco, A.E., E. Dal Bo, P.D. Ghiringhelli, C. Medrano, V. Romanowski,
A.N. Sarachu and O. Grau, 1989. Cloned cDNA probes for the
detection of Tomato spotted wilt virus. Phytopathol., 79: 1309-1313.
Roselló, S., M.J. Díez and F. Nuez, 1996. Viral diseases causing the
greatest economic losses to the tomato crop. I. the Tomato spotted
wilt virus - a revi. Scientia Hort., 67: 117-150.
Roossinck, M.J. 1997. Mechanisms of plant virus evolution. Ann. Rev.
Phytopathol., 35: 191-209.
80
Molecular biology of Tomato spotted wilt virus: An update
Rudolph, C., P.H. Schreier and J.F. Uhrig, 2003. Peptide-mediated
broad-spectrum plant resistance to tospoviruses. Proc. Natl. Acad.
Sci. USA., 100: 4429-34.
Sakimura, K. 1962. The present status of thrips borne viruses. In:
Biological Transmission of Disease Agents. K. Maramorosch (ed.).
Academic Press, New York, USA, 33-40.
Saldarelli, P., L. Barbarossa, F. Grieco and D. Gallitelli, 1996.
Digoxigenin- labelled riboprobes applied to phytosanitary
certification of tomato in Italy. Plant Dis., 80: 1343-1346.
Samuel, G., J.G. Bald and H.A. Pitman, 1930. Spotted wilt of tomatoes.
J. Austral. Science Ind. Res., 44.
Schnettler, E., H. Hemmes, R. Huismann, R. Goldbach, M. Prins and R.
Kormelink, 2010. Diverging affinity of Tospovirus RNA silencing
suppressor proteins, NSs, for various RNA Duplex Molecules. J.
Virol., 84: 11452-11554.
Scott, A., 2000. Tomato spotted wilt virus - positive steps towards
negative success. Mol. Plant Pathol., 1: 151-157.
Silva, M.S., C.R.F. Martins, I.C. Bezerra, T. Nagata, A.C. de Avila and
R.O. Resende, 2001. Sequence diversity of NSm movement protein
of Tospoviruses. Arch. Virol., 146: 1267-1281.
Sivparsad, B.J and A. Gubba, 2008. Isolation and molecular
characterization of Tomato spotted wilt virus (TSWV) isolates
occurring in South Africa. Afri. J. Agr. Res., 3: 428-434.
Snippe, M., J.W. Borst, R. Goldbach and R. Kormelink, 2007. Tomato
spotted wilt virus Gc and N proteins interact in vivo. Virol., 357:
115-123.
Snippe, M., J.W. Borst, R. Goldbach and R. Kormelink, 2005. The use
of fluorescence microscopy to visualize homotypic interactions of
Tomato spotted wilt virus nucleocapsid protein in living cells. J.
Virol. Methods., 125: 15-22.
Sin, S.H., B.C. McNulty, G.G. Kennedy and J.W. Moyer, 2005. Viral
genetic determinants for thrips transmission of Tomato spotted wilt
virus. Proc. Natl. Acad. Sci. USA., 102: 5168-73.
Soellick, T.R., J.F. Uhrig, G.L. Bucher, J.W. Kellmann and P.H. Schreier,
2000. The movement protein NSm of Tomato spotted wilt virus
(TSWV): RNA binding, interaction with the TSWV N protein and
identification of interacting proteins. Proc. Natl. Acad. Sci. USA.,
97: 2373- 2378.
Stafford, C.A., G.P. Walker and D.E. Ullman, 2010. Tomato spotted wilt
virus (Bunyaviridae, Tospovirus) infection alters feeding behavior
of its vector Frankliniella occidentalis (Pergande). Phytopathol.,
100: 122.
Stephens, E.B. and R.W. Compans, 1988. Assembly of animal viruses at
cellular membranes. Annu. Rev. Microbiol., 42: 489- 516.
Storms, M.M., R. Kormelink, D. Peters, J.W. Van Lent and R.W.
Goldbach, 1995. The nonstructural NSm protein of Tomato spotted
wilt virus induces tubular structures in plant and insect cells. Virol.,
214: 485-493.
Storms, M.M., T. Nagata, R. Kormelink, R.W. Goldbach and J.W.M. van
Lent, 2002. Expression of the movement protein of Tomato spotted
wilt virus in its insect vector Frankliniella occidentalis. Arch. Virol.,
147: 825-31.
Takeda, A., K. Sugiyama, H. Nagano, M. Mori, M. Kaido, K. Mise, S.
Tsuda and T. Okuno, 2002. Identification of a novel RNA silencing
suppressor, NSs protein of Tomato spotted wilt virus. FEBS Letters.,
532: 75-79.
Tsompana, M., J. Abad., M. Purugganan and J.W. Moyer, 2005. The
molecular population genetics of the Tomato spotted wilt virus
(TSWV) genome. Mol. Ecol., 14: 53-66.
Tsompana, M., J.W. Moyer, 2008. Tospoviruses. In: Encyclopedia of
Virology. Vol 5, B.W.J. Mahy, M.H.V. Van Regenmortel (eds.).
Elsevier, Oxford, UK. pp 157- 162.
Uhrig, J.F., T.R. Soellick, C.J. Minke, C. Philipp, J.W. Kellman and
J.W. Schreier, 1999. Homotypic interaction and multimerization of
nucleocapsid protein of Tomato spotted wilt tospovirus: identification
and characterization of two interacting domains. Proc. Natl. Acad.
Sci. USA., 96: 55- 60.
Ullman, D.E., J.L. Sherwood and T.L. German, 1997. In:Thripsas Crop
Pests. Thrips as vectors of plant pathogens. S.T. Lewis (ed.). CAB
International, Wallingford. pp. 539-565.
Ullman, D.E., A.E. Whitfield and T.L. German, 2005. Thrips and
tospoviruses come of age: mapping determinants of insect
transmission. Proc. Natl. Acad. Sci. USA., 102: 4931-4932.
Ullman, D.E., T.L. German, J.L. Sherwood, D.M. Westcot and F.A.
Cantone, 1993. Tospovirus replication in insect vector cells:
immunocytochemical evidence that the nonstructural protein
encoded by the S RNA of Tomato spotted wilt tospovirus is present
in thrips vector cells. Phytopathol., 83: 456-463.
Van Kammen, A., S. Henstra and T.S. Ie, I966. Morphology of Tomato
spotted wilt virus. Virol., 30: 574- 577.
van Knippenberg, I.C. 2005. Analysis of Tomato spotted wilt virus
Genome Transcription. PhD. Thesis, Wageningen University.
van Knippenberg, I., R. Goldbach and R. Kormelink, 2004. In vitro
transcription of Tomato spotted wilt virus is independent of
translation. J. Gen. Virol., 85: 1335-8.
van Knippenberg, I., M. Lamine, R. Goldbach and R. Kormelink, 2005.
Tomato spotted wilt virus transcriptase in vitro displays a preference
for cap donors with multiple base complementarity to the viral
template. Virol., 335: 122-130.
Van Poelwijk, F. 1996. On the expression strategy of the tospoviral genome.
Ph.D. Thesis, University of Wageningen, The Netherlands.
Van Poelwijk , F., K. Boye, R. Oosterling, D. Peters and R. Goldbach,
1993. Detection of the L protein of Tomato spotted wilt virus. Virol.,
197: 468-470.
Van Poelwijk, F., J. Kolkman and R. Goldbach, 1996. Sequence analysis
of the 5´ ends of Tomato spotted wilt virus N mRNAs. Arch. Virol.,
141: 177-184.
Van Regenmortel, M.H.V., C.M. Fauquet, D.H.L. Bishop, E.B. Carstens,
M.K. Estes, S. M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch,
C.R. Pringle and R.B. Wickner, 2000. In Seventh Report of the
International Committee on Taxonomy of Viruses. Academic Press,
San Diego CA, 1- 1162.
Webb, S.E., M.L. Kok-Yokomi and J.H. Tsai, 1997. Evaluation of
Frankliniella bispinosa as a potential vector of Tomato spotted wilt
virus. Phytopathol., 87:102.
Weekes, R., I. Barker and K.R. Wood, 1996. An RT-PCR test for the
detection of Tomato spotted wilt virus incorporating immunocapture
and colorimetric estimation. J. Phytopathol., 144: 575-580.
Whitfield, A.E., D.E. Ullman and T.L. German, 2005. Tomato spotted
wilt virus glycoprotein G(C) is cleaved at acidic pH. Virus Res., 110:
183-6.
Whitfield, A.E., N.K.K. Kumar, D. Rotenberg, D.E. Ullman, E.A.
Wyman, C .Zietlow, D.K. Willis and T.L. German, 2008. A soluble
form of the Tomato spotted wilt virus (TSWV) glycoprotein GN
(GN-S) inhibits transmission of TSWV by Frankliniella occidentalis.
Phytopathol., 98: 45-50.
Wijkamp, I. 1996. Virus-Vector Relationships in the Transmission of
Tospoviruses. Ph.D. Thesis, University of Wageningen, Wageningen,
The Netherlands.
Wijkamp, I., R. Almarza, R. Goldbach and D. Peters, 1995. Distinct levels
of specificity in thrips transmission of Tospovirus. Phytopathol., 85:
1069-1074.
Wijkamp, I., J.V. Lent, R. Kormelink, R. Goldbach and D. Peters, 1993.
Multiplication of Tomato spotted wilt virus in its insect vector
Frankliniella occidentalis. J. Gen. Virol., 74: 341- 349.
Received: January, 2013; Revised: February, 2013; Accepted: March, 2013